H]-DA release was decreased by blocking the spontaneous firing activity of DA neurons

Figure S1. Purification of DA neurons by cell sorting. Mesencephalic cell suspensions obtained from P14 mice were subjected to fluorescent-activated cell sorting. The fluorescent profile of WT (A) and TH-GFP (B) mice is shown. Note that no phycoerythrin labeling was used; the PEA filter was used to evaluate autofluorescence. Bidimensional plotting of GFP (fluorescent marked cells) vs PEA (autofluorescent cells) allowed us to discard cells that might have otherwise been collected using unidimensional sorting (only GFP) because of their high autofluorescence (more than 1000 fluorescent units, blue cell population). GFP-expressing DA neurons (P7 fraction) were sorted, their RNA extracted and subjected to RT-PCR (C). The expression of TH together with the absence of GAD-67 or GFAP (markers for GABA neurons and glía respectively) confirms the high purity level of these DA neuron preparations. -actin expression was evaluated as a positive control for the presence of mRNA. FACS, FACS purified DA neurons; M, RNA extracted from whole mesencephalon was used as a positive control; -, water, used as internal control. D) Potassium-evoked [ 3 H]-labeled DA release was quantified following radioactive labeling in the presence of clorgyline and pargyline. Labeling was performed either in the presence (KS) or absence (K) of the D2 autoreceptor antagonist sulpiride. When labeling occurred in the presence of sulpiride, extensive potassium-evoked [ 3 H]-labeled DA release was detected (left column). When labeling was performed in the absence of sulpiride (right column), little potassium-evoked release was detectable, most likely due to strong inhibition of TH activity by the D2 autoreceptor. The residual labeling and release seen in the absence of sulpiride is likely to represent [ 3 H]-labeled tyrosine metabolites, whereas release detected following labeling in the presence of sulpiride likely represents DA produced de novo by DA neurons using [2, 3, 5, 63 H]-Tyrosine. n=6. In each experiment, release measured in the group treated with sulpiride was normalized relative to release detected in the group not treated with sulpiride (K), which was set to 100%. * p<0.05. Figure S2. Syt7 expression and basic characteristics of DA release in culture. A) Cultured DA neurons were examined for Syt7 expression by immunocytochemistry. In addition to being found in the STD compartment (see Fig. 2), Syt7 (red signal, left panel) was also found to be localized in TH-positive (green signal, middle panel) axonal-like varicosities established by DA neurons. The right panel shows at higher magnification the colocalization of TH with Syt7 in the area identified by the white box in the central panel. B-F) The characteristics of DA release in rat cultures have been established previously; here we validate our radioassay method in mouse cultures. B) A decrease of extracellular Ca 2+ from 2 to 0.5 mM reduced but did not block the release of [ 3 H]-DA. In the presence of 0.5 mM extracellular Ca 2+ , conditions in which DA is released only from the somatodendritic compartment, [ 3 H]-DA release was decreased by blocking the spontaneous firing activity of DA neurons with 0.5 M TTX, (C) and was increased when cells were depolarized with 40 mM KCl (D). (E) Blockade of the DA transporter with 1 M GBR1209 increased the extracellular levels of somatodendritically-released DA. Numbers inside the bars indicate the number of experiments performed in each group. * p<0.05 versus control group. Error bars represent SD. (F) Glutamate-mediated synaptic currents were recorded from GFP-expressing DA neurons using the patch-clamp technique. In normal saline (2 mM extracellular Ca 2+ , top trace) numerous large amplitude synaptic currents were detected. In the presence of 0.5 mM extracellular Ca 2+ (lower trace), such events disappeared completely, leaving only occasional action potential-independent miniature synaptic currents. The bottom graph summarizes the results of 9 recorded neurons. In the presence of 0.5 mM extracellular Ca 2+ , all remaining events were miniature synaptic events of small amplitude.